Luciferases are enzymes that emit light in reaction with a specific substrate in the presence of co-factors. A diverse group of organisms use luciferase-mediated bioluminescence to startle predators or to attract prey or mates. The emitted light is used as a detection system for luciferase expression, which acts as a “reporter” for the activity of any regulatory element that controls its expression. Luciferase is particularly useful as a reporter enzyme in living cells and organisms. Firefly luciferase is one of many sensitive luciferases and is widely used by researchers to identify different biological events of cells in culture and in living small animals, and it is also used by public health researchers for the detection of food contamination.
The gene encoding firefly luciferase, cloned in 1985 from the North American firefly, Photinus pyralis is now emerging as the gene of choice for in vivo and in vitro reporting of transcriptional activity in eukaryotic cells. Reporter genes encoding for proteins with optical signatures, either fluorescent or bioluminescent, are a low-cost alternative for real-time analysis of gene expression in small animal models. In fluorescent approaches an external source of light is required for excitation of the protein. In contrast, bioluminescent reporter proteins can only produce light by using the appropriate substrates. Recently, several technical advances in utilizing highly sensitive detection devices have led to the biological use of cooled charge-coupled device (CCD) cameras capable of imaging very low levels of visible light emitted from internal organs of rodents. “Luciferase” is a family of photo-proteins that can be isolated from a large variety of insects, marine organisms, and prokaryotes. The emission spectrum ranges between 400 nm and 620 nm.
Functional proteins are made up of one or more polypeptides. Monomeric functional proteins can be split into two portions with resulting functionally inactive fragments. These split reporters have been used for measuring real time protein-protein interactions efficiently, both in cells and also in living animals. The inactive protein fragment assisted complementation of dihydrofolate reductase and β-lactamase have been used for studying protein-protein interactions in bacteria and mammalian cells. In previous studies, split bioluminescent monomeric proteins such as firefly luciferase and synthetic renilla luciferase, and the resulting fragments were used for studying protein-protein interactions. This was demonstrated by studying two known positive interacting proteins Id and myoD, and also by small molecule Rapamycin mediated interaction of human proteins FRB and FKBP12 in both cell culture and with noninvasive repetitive bioluminescence optical imaging in living mice. For many proteins, the backbones of the polypeptide chain have been split by chemical, proteolytic or genetic means and have created inter-chain packed active functional proteins. This combination of two protein fragments to restore activity has been termed as protein fragment complementation. The generation of functional proteins from a monomeric form to a heterodimeric form has been hypothesized as the phenomenon of the reversion of evolutionary process in which functional structures or domains are recruited and then fused at the genetic level. Even though many studies have reported the use of fragment-assisted complementation of optical reporter proteins for studying protein-protein interactions, we are not aware of any reports on the self-complementation of any of these reporters.